Vaccines for serogroup X meningococcus

A method for immunizing a subject against serogroup X meningococcus by administering a vaccine comprising one, two or all three of: (i) a meningococcal fHbp antigen; (ii) a meningococcal NHBA antigen; and/or (iii) a meningococcal NadA antigen. The vaccine may also include meningococcal outer membrane vesicles.

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Description
CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase of International Patent Application No. PCT/IB2013/054886 filed Jun. 14, 2013 and published in English, which claims the benefit of U.S. Provisional Application No. 61/659,595, filed Jun. 14, 2012. The entire contents of the foregoing applications are incorporated herein by reference for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 2, 2015, is named PAT055127-US-PCT_SL.txt and is 37,851 bytes in size.

TECHNICAL FIELD

This invention is in the field of vaccines for immunising against Neisseria meningitidis serogroup X.

BACKGROUND ART

Nearly all meningococcal disease is due to strains in serogroups A, B, C, W135 and Y, but serogroup X is also sometimes relevant. There is currently no vaccine for use against serogroup X. Thus there remains a need for a vaccine which would be effective against strains in serogroup X.

DISCLOSURE OF THE INVENTION

The BEXSERO® product (described in references 1 to 4; also known as 4CMenB) has been designed to immunise against serogroup B meningococcus. The inventors have found that patients immunised with BEXSERO® are also protected against strains in serogroup X.

Thus the invention provides a method for immunising a subject against serogroup X meningococcus by administering an immunogenic composition comprising one, two or three of: (i) a meningococcal fHbp antigen; (ii) a meningococcal NHBA antigen; and/or (iii) a meningococcal NadA antigen.

Similarly, the invention provides an immunogenic composition for use in immunising a subject against serogroup X meningococcus, wherein the immunogenic composition comprises one, two or three of: (i) a meningococcal fHbp antigen; (ii) a meningococcal NHBA antigen; and/or (iii) a meningococcal NadA antigen.

Also, the invention provides the use of one, two or three of: (i) a meningococcal fHbp antigen; (ii) a meningococcal NHBA antigen; and/or (iii) a meningococcal NadA antigen, in the manufacture a medicament for immunising a subject against serogroup X meningococcus.

In some embodiments the immunogenic composition also includes meningococcal outer membrane vesicles; in other embodiments the immunogenic composition is free from meningococcal outer membrane vesicles. Where the composition includes meningococcal outer membrane vesicles then the immunogenic composition includes at least one of the fHbp, NHBA and/or NadA antigen(s) in non-OMV form e.g. in soluble form.

Serogroup X Protection

The invention is used to immunise subjects against serogroup X meningococcus, such that recipients of the immunogenic composition mount an immune response which provides protection against infection by and/or disease due to Neisseria meningitidis bacteria in serogroup X. This serogroup is characterised by a capsular saccharide having chains of (α1→4)-linked N-acetylglucosamine 1-phosphate.

Protection against serogroup X strains can be measured epidemiologically, but it is more common and convenient to use an indirect measure such as to confirm that an immunogenic composition elicits a serum bactericidal antibody (SBA) response in recipients. The SBA assay is standard in this field (e.g. see references 5-8) and it shows good inter-laboratory reproducibility when using harmonised procedures [9]. In brief, sera from recipients of the composition are incubated with target bacteria (in the present invention, serogroup X meningococci) in the presence of complement (preferably human complement, although baby rabbit complement is often used instead) and killing of the bacteria is assessed at various dilutions of the sera to determine SBA activity.

It is not necessary that the composition should protect against each and every strain of serogroup X meningococcus, or that each and every recipient of the composition must be protected. Such universal protection is not the normal standard in this field. Rather, protection is normally assessed against a panel of clinically-relevant isolates, often selected on a country-by-country basis and perhaps varying with time, and is measured across a population of recipients. Various serogroup X strains are available for confirming protective efficacy e.g. the composition might protect against M405 (NAMRU#4; ATCC 35560), against strain 860060 (reference strain 657 from the PubMLST database; strain designation X:P1.12-1, 13-5:F5-5: ST-24 (cc750); also known as Z6430), against strains 9557, 9558 &/or 9559 [30], against the serogroup X strains listed in Table 1 of ref 10, against the strains characterised in reference 11, etc.

Within serogroup X, when the immunogenic composition includes a fHbp antigen, the method may be useful for immunising against strains or isolates having the same fHbp variant as the variant in the composition e.g. if the composition includes a variant 1 fHbp then the method will be most useful for immunising against serogroup X strains which express a variant 1 fHbp.

As well as being immunised against serogroup X meningococcus, recipients may also be immunised against other serogroups e.g. one or more of serogroups A, B, C, W135 and/or Y. For instance, reference 12 reports that the antigens in BEXSERO® can protect against serogroup Y, and reference 13 suggests that fHbp might provide protection beyond serogroup B alone.

The Immunogenic Composition

The invention uses an immunogenic composition (e.g. a vaccine) to protect subjects against serogroup X meningococci. The composition includes at least one of fHbp, NHBA and/or NadA antigens, and it elicits an immune response against these included antigens. In some embodiments the composition includes only one of these three antigens (but may include further antigens) e.g. fHBP, NHBA or NadA. In some embodiments the composition includes only two of these three antigens (but may include further antigens) e.g. fHBP+NHBA, fHBP+NadA, NHBA+NadA. In other embodiments the composition includes all three of these three antigens (and may include further antigens).

The composition does not include an immunogenic amount of serogroup X capsular saccharide i.e. protection against serogroup X cannot be explained by an anti-saccharide response. Serogroup X capsular saccharide is absent as free saccharide, conjugated saccharide, or membrane-located saccharide (e.g. in OMVs).

A preferred composition includes each of: (i) a fHbp antigen comprising amino acid sequence SEQ ID NO: 6 e.g. SEQ ID NO: 7; (ii) a NHBA antigen comprising amino acid sequence SEQ ID NO: 8 SEQ ID NO: 9; and (iii) a NadA antigen comprising amino acid sequence SEQ ID NO: 10. BEXSERO® is one such composition.

Although SEQ ID NOs: 6, 8 and 10 are useful amino acid sequences in a combination, the invention is not limited to these precise sequences. Thus 1, 2, or all 3 of these amino acid sequences can independently be modified by up to 5 single amino changes (i.e. 1, 2, 3, 4 or 5 single amino acid substitutions, deletions and/or insertions) provided that the modified sequence can elicit antibodies which still bind to a polypeptide consisting of the unmodified sequence.

The polypeptides in a composition may be present at substantially equal masses i.e. the mass of each of them is within +5% f the mean mass of all the polypeptides. Thus, where the composition includes three polypeptides, one for each of fHbp, HNBA and NadA, they may be present at a mass ratio of a:b:c, where each of a, b & c is between 0.95 and 1.05.

fHbp (Factor H Binding Protein)

The fHbp antigen has been characterised in detail. It has also been known as protein ‘741’ (SEQ IDs 2535 & 2536 in ref. 26), ‘NMB1870’, ‘GNA1870’ [14-16], ‘P2086’, ‘LP2086’ or ‘ORF2086’ [17-19], It is naturally a lipoprotein and is expressed across many meningococcal serogroups. The structure of fHbp's C-terminal immunodominant domain (‘fHbpC’) has been determined by NMR [20], This part of the protein forms an eight-stranded β-barrel, whose strands are connected by loops of variable lengths. The barrel is preceded by a short α-helix and by a flexible N-terminal tail. The protein was confirmed as a factor H binding protein, and named fHbp, in reference 21.

The fHbp antigen falls into three distinct variants [22] and it has been found that serum raised against a given family is bactericidal within the same family, but is not active against strains which express one of the other two families i.e. there is intra-family cross-protection, but not inter-family cross-protection. The invention can use a single fHbp variant, but to provide broader coverage a composition can usefully include a fHbp from two or three of the variants.

Where a composition comprises a single fHBP antigen it may include one of the following:

    • (a) a first polypeptide comprising a first amino acid sequence, where the first amino acid sequence comprises an amino acid sequence (i) having at least a % sequence identity to SEQ ID NO: 1 and/or (ii) consisting of a fragment of at least x contiguous amino acids from SEQ ID NO: 1;
    • (b) a second polypeptide, comprising a second amino acid sequence, where the second amino acid sequence comprises an amino acid sequence (i) having at least b % sequence identity to SEQ ID NO: 2 and/or (ii) consisting of a fragment of at least y contiguous amino acids from SEQ ID NO: 2;
    • (c) a third polypeptide, comprising a third amino acid sequence, where the third amino acid sequence comprises an amino acid sequence (i) having at least c % sequence identity to SEQ ID NO: 3 and/or (ii) consisting of a fragment of at least z contiguous amino acids from SEQ ID NO: 3.

Where a composition comprises two different meningococcal fHBP antigens, it may include a combination of: (i) a first and second polypeptide as defined above; (ii) a first and third polypeptide as defined above; or (iii) a second and third polypeptide as defined above. A combination of a first and third polypeptide is preferred.

In other embodiments a composition comprises three different meningococcal fHBP antigens, with first, second and third polypeptides as defined above.

Where a composition comprises two or three different meningococcal fHBP antigens, although these may share some sequences in common, the first, second and third polypeptides have different fHBP amino acid sequences.

A polypeptide comprising the first amino acid sequence will, when administered to a subject, elicit an antibody response comprising antibodies that bind to the wild-type meningococcus protein having mature amino acid sequence SEQ ID NO: 1 (strain MC58). In some embodiments some or all of these antibodies do not bind to the wild-type meningococcus protein having mature amino acid sequence SEQ ID NO: 2 or to the wild-type meningococcus protein having mature amino acid sequence SEQ ID NO: 3.

A polypeptide comprising the second amino acid sequence will, when administered to a subject, elicit an antibody response comprising antibodies that bind to the wild-type meningococcus protein having mature amino acid sequence SEQ ID NO: 2 (strain 961-5945). In some embodiments some or all of these antibodies do not bind to the wild-type meningococcus protein having mature amino acid sequence SEQ ID NO: 1 or to the wild-type meningococcus protein having mature amino acid sequence SEQ ID NO: 3.

A polypeptide comprising the third amino acid sequence will, when administered to a subject, elicit an antibody response comprising antibodies that bind to the wild-type meningococcus protein having mature amino acid sequence SEQ ID NO: 3 (M1239). In some embodiments some or all of these antibodies do not bind to the wild-type meningococcus protein having mature amino acid sequence SEQ ID NO: 1 or to the wild-type meningococcus protein having mature amino acid sequence SEQ ID NO: 2.

In some embodiments the fragment of at least x contiguous amino acids from SEQ ID NO: 1 is not also present within SEQ ID NO: 2 or within SEQ ID NO: 3. Similarly, the fragment of at least y contiguous amino acids from SEQ ID NO: 2 might not also be present within SEQ ID NO: 1 or within SEQ ID NO: 3. Similarly, the fragment of at least z contiguous amino acids from SEQ ID NO: 3 might not also be present within SEQ ID NO: 1 or within SEQ ID NO: 2. In some embodiments, when said fragment from one of SEQ ID NOs: 1 to 3 is aligned as a contiguous sequence against the other two SEQ ID NOs, the identity between the fragment and each of the other two SEQ ID NOs is less than 75% e.g. less than 70%, less than 65%, less than 60%, etc.

The value of a is at least 80 e.g. 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or more. The value of b is at least 80 e.g. 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or more. The value of c is at least 80 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or more. The values of a, b and c may be the same or different. In some embodiments, a b and c are identical.

The value of x is at least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250). The value of y is at least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250). The value of z is at least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250). The values of x, y and z may be the same or different. In some embodiments, iv and z are identical.

Fragments preferably comprise an epitope from the respective SEQ ID NO: sequence. Other useful fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of the respective SEQ ID NO: while retaining at least one epitope thereof.

Amino acid sequences used with the invention may, compared to SEQ ID NOs: 1 2 or 3, include one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) conservative amino acid replacements i.e. replacements of one amino acid with another which has a related side chain. Genetically-encoded amino acids are generally divided into four families: (1) acidic i.e. aspartate, glutamate; (2) basic i.e. lysine, arginine, histidine; (3) non-polar i.e. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar i.e. glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In general, substitution of single amino acids within these families does not have a major effect on the biological activity. The polypeptides may have one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) single amino acid deletions relative to a reference sequence. The polypeptides may also include one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) insertions (e.g. each of 1, 2, 3, 4 or 5 amino acids) relative to a reference sequence.

A useful first amino acid sequence has at least 85%) identity (e.g. ≥95% or 100%) to SEQ ID NO: 1. Another useful first amino acid sequence has at least 95% identity (e.g. ≥98%) or 100%) to SEQ ID NO: 12.

A useful third amino acid sequence has at least 85% identity (e.g. ≥95% or 100%) to SEQ ID NO: 3. Another useful third amino acid sequence has at least 95% identity (e.g. ≥98% or 100%) to SEQ ID NO: 11.

Combinations comprising a mixture of first and third sequences based around SEQ ID NOs: 11 and 12 (or their close variants) are particularly useful. Thus a composition may comprise a polypeptide comprising amino acid sequence SEQ ID NO: 11 and a further polypeptide comprising amino acid sequence SEQ ID NO: 12.

Another useful fHbp which can be used with the invention is one of the modified forms disclosed, for example, in reference 23 e.g. comprising SEQ ID NO: 20 or 23 therefrom. These modified forms can use a single fHbp polypeptide to elicit antibody responses which are broadly bactericidal against various fHbp variants. SEQ ID NO: 77 in reference 23 is another useful fHbp sequence which can be used.

fHbp antigens used with the invention can be lipidated e.g. at a N-terminus cysteine residue. In other embodiments they will not be lipidated, and may include amino acid sequences upstream of the natural mature N-terminal cysteine. SEQ ID NOs: 1-3 and 11-12 begin with the cysteine from the natural N-terminus of the relevant mature fHbp polypeptides. For lipidated fHBPs, lipids attached to cysteines will usually include palmitoyl residues e.g. as tripalmitoyl-S-glyceryl-cysteine (Pam3Cys), dipalmitoyl-S-glyceryl cysteine (Pam2Cys), N-acetyl (dipalmitoyl-S-glyceryl cysteine), etc.

Administration of a fHBP will preferably elicit antibodies which can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 1, 2 or 3. Advantageous fHBP antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject.

The total amount of a fHBP polypeptide will usually be between 1 and 500 μg/dose e.g. between 60 and 200 μg/dose or between 120 and 500 μg/ml.

A polypeptide including the fHbp antigen sequence can include that sequence alone, or it can be a fusion polypeptide. One useful fusion partner for a fHbp sequence is the NMB2091 polypeptide, which will normally be upstream of the fHbp sequence. Thus the fHbp antigen can be present in a composition of the invention as a NMB2091-fHbp fusion e.g. SEQ ID NO: 7.

NHBA (Neisserial Heparin Binding Antigen)

NHBA was included in the published genome sequence for meningococcal serogroup B strain MC58 [24] as gene NMB2132 (GenBank accession number GI:7227388; SEQ ID NO: 4 herein). Sequences of NHBA from many strains have been published since then. For example, allelic forms of NHBA (referred to as protein ‘287’) can be seen in FIGS. 5 and 15 of reference 25, and in example 13 and FIG. 21 of reference 26 (SEQ IDs 3179 to 3184 therein). Various immunogenic fragments of NHBA have also been reported. The protein was confirmed as a heparin binding protein, and named NHBA, in reference 27.

Preferred NHBA antigens for use with the invention comprise an amino acid sequence: (a) having 70% r more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% r more) to SEQ ID NO: 4; and/or (b) comprising a fragment of at least ‘n’ consecutive amino acids of SEQ ID NO: 4, wherein ‘n’ is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 4.

The most useful NHBA antigens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 4. Advantageous NHBA antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject.

A polypeptide including the NHBA antigen sequence can include that sequence alone, or it can be a fusion protein. One useful fusion partner for a NHBA sequence is the NMB1030 polypeptide, which will normally be downstream of the NHBA sequence. Thus the NHBA antigen can be present in a composition of the invention as a NHBA-NMB1030 fusion e.g. SEQ ID NO: 9.

NadA (Neisserial Adhesin A)

The NadA antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [24] as gene NMB1994 (GenBank accession number GI:7227256; SEQ ID NO: 5 The sequences of NadA antigen from many strains have been published since then, and the protein's activity as a Neisserial adhesin has been well documented. Various immunogenic fragments of NadA have also been reported. The protein was confirmed as an adhesin, and named NadA, in reference 28.

Preferred NadA antigens for use with the invention comprise an amino acid sequence: (a) having 70% r more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% r more) to SEQ ID NO: 5; and/or (b) comprising a fragment of at least ‘n’ consecutive amino acids of SEQ ID NO: 5, wherein ‘n’ is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 5.

The most useful NadA antigens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 5. Advantageous NadA antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject. SEQ ID NO: 10 is one such fragment.

Outer Membrane Vesicles

In some embodiments the immunogenic composition is free from meningococcal outer membrane vesicles (OMVs). In other embodiments, however, the immunogenic composition includes meningococcal OMVs. In such OMV-containing embodiments the composition includes at least one of fHbp, NHBA and/or NadA antigen in non-OMV form e.g. in soluble form. Thus these compositions are prepared by mixing OMVs with one or more soluble antigen(s), which contrasts with the approach taken in references 29 and 30.

Where a composition includes OMVs, these OMVs can be any proteoliposomic vesicle obtained by disruption of or blebbing from a meningococcal outer membrane to form vesicles therefrom that retain antigens from the outer membrane. Thus this term includes, for instance, OMVs (sometimes referred to as ‘blebs’), microvesicles (MVs) and ‘native OMVs’ (‘NOMVs’). Various such vesicles are known in the art (e.g. see references 31 to 45) and any of these can be included within a composition of the invention.

Further Meningococcal Antigens

A composition can include one or more further meningococcal protein antigens, such as HmbR, NspA, NhhA, App, Omp85, TbpA, TbpB, and/or Cu,Zn-superoxide dismutase.

A composition can include one or more meningococcal saccharide antigens, which will typically be conjugated to carrier proteins. Thus, for instance, a composition might include one or more capsular saccharides from serogroups A, C, W135 and/or Y. For instance, the composition might include the conjugates which are present in the MENVEO, MENACTRA, or NIMENRIX products (all of which include conjugated capsular saccharides for each of serogroups A, C, W135 and Y).

Non-Meningococcal Antigens

A composition can include one or more non-meningococcal antigens. For instance, it can include one or more of. (a) an antigen from Streptococcus pneumoniae, such as a saccharide (typically conjugated), as in the PREVNAR and SYNFLORIX products; (b) an antigen from hepatitis B virus, such as the surface antigen HBsAg; (c) an antigen from Bordetella pertussis, such as pertussis holotoxin (PT) and filamentous haemagglutinin (FHA) from B. pertussis, optionally also in combination with pertactin and/or agglutinogens 2 and 3; (d) a diphtheria antigen, such as a diphtheria toxoid; (e) a tetanus antigen, such as a tetanus toxoid; (f) a saccharide antigen from Haemophilus influenzae B (Hib), typically conjugated; and/or (g) inactivated poliovirus antigens.

Non-Antigen Components

In addition to its antigens, an immunogenic composition of the invention typically includes a pharmaceutically acceptable carrier, and a thorough discussion of such carriers is available in reference 46.

The pH of a composition is usually between 6 and 8, and more preferably between 6.5 and 7.5 (e.g. about 7). Stable pH may be maintained by the use of a buffer e.g. a Tris buffer, a citrate buffer, phosphate buffer, or a histidine buffer. Thus a composition will generally include a buffer.

A composition may be sterile and/or pyrogen-free. Compositions may be isotonic with respect to humans.

A composition comprises an immunologically effective amount of its antigen(s). An ‘immunologically effective amount’ is an amount which, when administered to a subject, is effective for eliciting an antibody response against the antigen. This amount can vary depending upon the health and physical condition of the individual to be treated, their age, the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. The antigen content of compositions of the invention will generally be expressed in terms of the mass of protein per dose. A dose of 10-500 μg (e.g. 50 μg) per antigen can be useful.

Immunogenic compositions may include an immunological adjuvant. Thus, for example, they may include an aluminium salt adjuvant or an oil-in-water emulsion (e.g. a squalene-in-water emulsion). Suitable aluminium salts include hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates), (e.g. see chapters 8 & 9 of ref. 47), or mixtures thereof. The salts can take any suitable form (e.g. gel, crystalline, amorphous, etc.), with adsorption of antigen to the salt being preferred. The concentration of Al+++ in a composition for administration to a patient is preferably less than 5 mg/ml e.g. ≤4 mg/ml, ≤3 mg/ml, ≤2 mg/ml, ≤1 mg/ml, etc. A preferred range is between 0.3 and 1 mg/ml. A maximum of 0.85 mg/dose is preferred. Aluminium hydroxide and aluminium phosphate adjuvants are particularly suitable for use with the invention.

Compositions may include an antimicrobial, particularly when packaged in multiple dose format. Antimicrobials such as thiomersal and 2-phenoxyethanol are commonly found in vaccines, but it is preferred to use either a mercury-free preservative or no preservative at all.

Compositions may comprise detergent e.g. a Tween (polysorbate), such as Tween 80. Detergents are generally present at low levels e.g. <0.01%. Compositions may include residual detergent (e.g. deoxycholate) from OMV preparation. The amount of residual detergent is preferably less than 0.4 μg (more preferably less than 0.2 μg) for every μg of meningococcal protein.

If a vaccine includes LOS, the amount of LOS is preferably less than 0.12 μg (more preferably less than 0.05 μg) for every μg of protein.

Compositions may include sodium salts (e.g. sodium chloride) to give tonicity. A concentration of 10±2 mg/ml NaCl is typical e.g. about 9 mg/ml.

Administration of the Composition

Compositions of the invention will generally be administered directly to a patient. Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or by any other suitable route. Intramuscular administration is preferred e.g. to the thigh or the upper arm. Injection may be via a needle (e.g. a hypodermic needle), but needle-free injection may alternatively be used. A typical intramuscular dosage volume is 0.5 ml.

Administration can involve a single dose schedule, but will usually involve a multiple dose schedule. Suitable intervals between priming doses can be routinely determined e.g. between 4-16 weeks, such as one month or two months. BEXSERO® can be administered at ages of 2, 4 & 6 months, or at 2, 3 & 4 months, with a fourth optional dose at 12 months.

The subject who is immunised is a human being, who may be any age e.g. 0-12 months old, 1-5 years old, 5-18 years old, 18-55 years old, or more than 55 years old.

General

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., references 48-54, etc.

The term “comprising” encompasses “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X±Y.

The term “about” in relation to a numerical value x is optional and means, for example, x±10%.

Where the invention concerns an “epitope”, this epitope may be a B-cell epitope and/or a T-cell epitope, but will usually be a B-cell epitope. Such epitopes can be identified empirically (e.g. using PEPSCAN [55,56] or similar methods), or they can be predicted (e.g. using the Jameson-Wolf antigenic index [57], matrix-based approaches [58], MAPITOPE [59], TEPITOPE [60,61], neural networks [62], OptiMer & EpiMer [63, 64], ADEPT [65], Tsites [66], hydrophilicity [67], antigenic index [68] or the methods disclosed in references 69-73, etc.). Epitopes are the parts of an antigen that are recognised by and bind to the antigen binding sites of antibodies or T-cell receptors, and they may also be referred to as “antigenic determinants”.

References to a percentage sequence identity between two amino acid sequences means that, when aligned, that percentage of amino acids are the same in comparing the two sequences. This alignment and % homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of ref. 74. A preferred alignment is determined by the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is disclosed in ref. 75.

The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.

BRIEF DESCRIPTION OF DRAWINGS

There are no drawings.

MODES FOR CARRYING OUT THE INVENTION

The BEXSERO® product is described in references 1 to 3 and it includes 50 μg of each of NadA (subvariant 3.1), fHbp subvariant 1.1 (as a GNA2091-fHbp fusion protein), and NHBA subvariant 1.2 (as a NHBA-GNA1030 fusion protein), adsorbed onto 1.5 mg aluminium hydroxide, and with 25 μg OMVs from N. meningitidis strain NZ98/254.

Eleven MenX strains, isolated between 1995 and 2007 from several countries, were obtained. Their serotype and serosubtype [76], MLST [77] and genotype were as follows:

Clonal PorA Strain Serotype Subtype complex VR1 VR2 FetA fHbp NHBA nadA LNP13407 Non typeable P1.5 ST-181 5-1 10-1 F4-23 1.60 358 − LNP14354 Non typeable P1.5 ST-181 5-1 10-1 F4-23 1.461 358 − LNP14355 Non typeable P1.5 ST-181 5-1 10-1 F4-23 1.461 358 − LNP14964 Non typeable P1.5 ST-181 5-1 10-1 F4-23 1.461 358 − LNP15038 Non typeable P1.5 ST-181 5-1 10-1 F4-23 1.461 358 − LNP15075 Non typeable P1.5 ST-181 5-1 10-1 F4-23 1.60 358 + LNP15877 Non typeable P1.5 ST-181 5-1 10-1 F4-23 1.60 358 − LNP23557 Non typeable P1.5 ST-181 5-1 10-1 F4-23 1.461 358 − LNP23558 Non typeable P1.5 ST-181 5-1 10-1 F4-23 1.61 359 − LNP24196 4 P1.12 None 12-1   16-52 F3-9 2.23 51 + LNP24287 4 P1.16 ST-750 21 16 F5-5 3.69 129 −

The first 9 strains, all non-typeable, in the same clonal complex, and having the same PorA variable region and FetA marker and fHbp subtype (variant 1), were from African countries; the other two strains were isolated in France.

For SBA, sera were obtained from clinical studies before vaccination and after the administration of the BEXSERO® vaccine. For infants, the tested samples were from 40 infants who had received either three immunizations or three immunizations plus one booster. For other age groups, pooled sera were from 12 adolescents or 23 adults vaccinated with two doses. Most of the adults had already received quadrivalent (ACWY) polysaccharide vaccine. A polyclonal rabbit serum against the serogroup X capsular saccharide was used as a positive control.

Human complement was used for the SBA assays, with serum from vaccinated subject (hSBA) and a polyclonal serum used as control. Protection was defined as a titre of 4 for hSBA [78], Vaccine response was scored either as the percentage of hSBA titres of at least 8 for strains that showed a response <4 prior to vaccination, or as 4 fold increase for strains that showed a response of at least 4 to vaccination. SBA titres were as follows:

Infants Positive Adults Adolescents Post- Strain control Pre- Post-3 Pre Post-2 Pre Post-3 4 LNP13407 >4096 4 >128 <4 128 <2 32 64 LNP14354 2048 16 >128 4 >128 <2 >64 >64 LNP14355 2048 8 64 4 >128 <2 >64 >64 LNP14964 2048 <4 >128 <4 >128 <2 32 >64 LNP15038 1024 16 >128 <4 >128 <2 >64 >64 LNP15075 128 4 128 <4 32 <2 16 16 LNP15877 2048 4 >128 8 >128 <2 >64 >64 LNP23557 2048 <4 128 <4 64 <2 16 32 LNP23558 >4096 16 >128 <4 >128 <4 16 64 LNP24196 128 <4 4 <4 4 <2 2 2 LNP24287 1024 <4 4 <4 8 <2 <2 2

Thus all isolates had SBA titres of ≥128 using the polyclonal anti-capsule serum. In infants, all pre-immunisation titers were lower than 8 (the hSBA titre which correlateds with protection)S. Slightly higher pre-immunisation titres were observed in adolescents and adults.

After vaccination hSBA titres increased in all tested schedules and age groups, against all isolates from Africa. For isolates with a titres of at least 4 before vaccination, a 4-fold increase in hSBA titres was observed in all cases except for the strain LNP14355 (3-fold increase). In contrast to the African strains, the two isolates from France were not killed by the post-immunisation sera, although SBA titers increased in all cases.

Thus the MenX strains involved in meningococcal meningitis (at least for African strains isolated between 1995-2007) can be covered by the 4CMenB BEXSERO® vaccine. Coverage of these isolates was well predicted based on their fHbp variant type.

It will be understood that the invention is described above by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

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Claims

1. A method for immunizing a subject in need thereof against serogroup X meningococcus by administering to the subject a composition comprising a meningococcal fHbp antigen and an aluminum salt adjuvant; wherein the meningococcal fHbp antigen comprises amino acid sequence SEQ ID NO: 7, wherein the composition either (a) is free from meningococcal outer membrane vesicles or (b) includes meningococcal outer membrane vesicles, and the fHbp antigen is present in the composition in soluble form, wherein the fHbp antigen is not conjugated to a meningococcal polysaccharide, and wherein the composition does not contain un-conjugated meningococcal polysaccharides.

2. The method of claim 1, wherein the composition further comprises one or both of (i) a meningococcal NHBA antigen, and (ii) a meningococcal NadA antigen.

3. The method of claim 2, wherein the composition includes the meningococcal outer membrane vesicles and the meningococcal NHBA antigen, the meningococcal NadA antigen, or both are present in the composition in soluble form.

4. The method of claim 2, wherein the meningococcal NHBA antigen comprises (a) an amino acid sequence at least 95% identical to SEQ ID NO: 4.

5. The method of claim 2, wherein the meningococcal NadA antigen comprises (a) an amino acid sequence at least 95% identical to SEQ ID NO: 5.

6. The method of claim 1, wherein the composition includes each of: (i) an antigen comprising amino acid sequence of SEQ ID NO: 8; and (ii) an antigen comprising amino acid sequence of SEQ ID NO: 10.

7. The method of claim 1, wherein the composition includes each of: (i) an antigen comprising amino acid sequence SEQ ID NO: 9; and (ii) an antigen comprising amino acid sequence SEQ ID NO: 10.

8. The method of claim 2, wherein the antigens are present at substantially equal masses.

9. The method of claim 1, wherein the composition further comprises one or more additional meningococcal protein antigens.

10. The method of claim 1, where the administration induces an immune response against one or more of meningococcal serogroups A, B, C, W135, and Y.

11. The method of claim 1, wherein the composition further comprises one or more non-meningococcal antigens.

12. The method of claim 1, wherein the administration is by intramuscular injection.

13. The method of claim 1, wherein the subject is a human and the human has (i) a 4 fold increase in human serum bactericidal antibody (hSBA) titer after administration if the subject had at least an hSBA titer of 4 before administration, or (ii) an hSBA titer of 8 after administration if the subject had an hSBA titer of less than 4 before administration.

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Patent History
Patent number: 10376573
Type: Grant
Filed: Jun 14, 2013
Date of Patent: Aug 13, 2019
Patent Publication Number: 20160166674
Assignees: GlaxoSmithKline Biologicals SA (Rixensart), Institut Pasteur (Paris)
Inventors: Mariagrazia Pizza (Siena), Peter Dull (Seattle, WA), Marzia Monica Giuliani (Siena), Muhamed-Kheir Taha (Saint Maur des Fosses), Eva Hong (Maisons-Alfort), Ala-Eddine Deghmane (Gagny)
Primary Examiner: Oluwatosin A Ogunbiyi
Application Number: 14/407,987
Classifications
Current U.S. Class: Non/e
International Classification: A61K 39/095 (20060101); A61K 39/39 (20060101); A61K 39/00 (20060101);